4. Load samples onto the gel. Run the electrophoresis at 150 V in
1Â TBE until the bromophenol blue band reaches the bottom
of the gel.
5. Prepare a container with water and add ethidium bromide to a
final concentration of 0.5 μg/L.
6. Separate the gel plates with a spatula. Carefully take off the gel
and transfer it to the ethidium bromide solution. Incubate for
5–10 min at room temperature with gentle agitation. Image
the RNA profile with a UV transilluminator.
3.9 Electrophoretic
Blotting Procedure
1. Cut four Whatman sheets to the size of the plates, and a
Hybond N+ membrane square to a size slightly larger than
the gel.
2. Soak a sponge from the electroblot apparatus with 0.5Â TBE
and place it in the opened electroblotting cassette. Wet two
sheets of Whatman paper with 0.5Â TBE and place them onto
the sponge. Soak the Hybond N+ membrane with 0.5Â TBE
and place it onto the top of the Whatman sheets. Rinse the gel
in 0.5Â TBE and transfer the gel onto the nylon membrane.
Remove any bubbles (see Note 15). Soak two more sheets of
Whatman paper and place them on top of the membrane. Wet
the second sponge with 0.5Â TBE and place it on top. Clamp
the cassette closed and place the assembled system into the
chamber. Pay attention to the cassette orientation in the chamber to allow transfer of RNAs from the gel onto the membrane.
Fill the chamber with 0.5Â TBE.
3. Carry out the transfer of the RNAs from the gel to the membrane at 300 mA for 80 min at 4
C.
4. Place two sheets of Whatman paper in a container and soak
them with 2Â SSC. Discard excessive buffer. Disassemble the
electroblotting system, remove the nylon membrane, and place
it onto the sheets of Whatman paper with the surface carrying
the RNA facing up. Incubate for 10 min at room temperature.
Place the container into a Stratalinker to cross-link the RNA
to the membrane by applying two UV exposures of
120,000 μJ/cm
2 .
3.10 Labeling of
the Oligodeoxynucleotide Probe
All steps requiring manipulation of radioactivity must be carried out
in an appropriate room. The safety rules of your institution must be
strictly followed.
1. Preheat a water bath at 37
C.
2. Mix 4.5 μL of water, 1 μL of 100 μM oligodeoxynucleotide
complementary to the small RNA of interest, 1 μL of 10Â T4
polynucleotide kinase (PNK) buffer, 1 μL of PNK, and 2.5 μL
of [γ
32
P]-ATP. Incubate for 30 min at 37
C.
Plant Protoplasts-Based System to Transfect RNA
425
1Â TBE until the bromophenol blue band reaches the bottom
of the gel.
5. Prepare a container with water and add ethidium bromide to a
final concentration of 0.5 μg/L.
6. Separate the gel plates with a spatula. Carefully take off the gel
and transfer it to the ethidium bromide solution. Incubate for
5–10 min at room temperature with gentle agitation. Image
the RNA profile with a UV transilluminator.
3.9 Electrophoretic
Blotting Procedure
1. Cut four Whatman sheets to the size of the plates, and a
Hybond N+ membrane square to a size slightly larger than
the gel.
2. Soak a sponge from the electroblot apparatus with 0.5Â TBE
and place it in the opened electroblotting cassette. Wet two
sheets of Whatman paper with 0.5Â TBE and place them onto
the sponge. Soak the Hybond N+ membrane with 0.5Â TBE
and place it onto the top of the Whatman sheets. Rinse the gel
in 0.5Â TBE and transfer the gel onto the nylon membrane.
Remove any bubbles (see Note 15). Soak two more sheets of
Whatman paper and place them on top of the membrane. Wet
the second sponge with 0.5Â TBE and place it on top. Clamp
the cassette closed and place the assembled system into the
chamber. Pay attention to the cassette orientation in the chamber to allow transfer of RNAs from the gel onto the membrane.
Fill the chamber with 0.5Â TBE.
3. Carry out the transfer of the RNAs from the gel to the membrane at 300 mA for 80 min at 4
C.
4. Place two sheets of Whatman paper in a container and soak
them with 2Â SSC. Discard excessive buffer. Disassemble the
electroblotting system, remove the nylon membrane, and place
it onto the sheets of Whatman paper with the surface carrying
the RNA facing up. Incubate for 10 min at room temperature.
Place the container into a Stratalinker to cross-link the RNA
to the membrane by applying two UV exposures of
120,000 μJ/cm
2 .
3.10 Labeling of
the Oligodeoxynucleotide Probe
All steps requiring manipulation of radioactivity must be carried out
in an appropriate room. The safety rules of your institution must be
strictly followed.
1. Preheat a water bath at 37
C.
2. Mix 4.5 μL of water, 1 μL of 100 μM oligodeoxynucleotide
complementary to the small RNA of interest, 1 μL of 10Â T4
polynucleotide kinase (PNK) buffer, 1 μL of PNK, and 2.5 μL
of [γ
32
P]-ATP. Incubate for 30 min at 37
C.
Plant Protoplasts-Based System to Transfect RNA
425